The overall objectives of this project are to ascertain the mechanisms by which the ovarian steroid 17 - estradiol (E2) regulates GnRH neuronal excitability, which controls GnRH neurosecretion and fertility. These neurons are located in the hypothalamus and constitute the final step in the regulation of pituitary luteinizing hormone (LH) secretion and ovulation in females. Importantly, E2 feeds back to alternately inhibit and stimulate GnRH neurosecretion, via negative and positive feedback, respectively. Extensive studies have demonstrated that these E2 actions are complex and involve multiple neurotransmitters and metabolic factors. However, we have limited knowledge about the cellular and molecular mechanisms by which GnRH neurons are regulated, and therefore, incomplete understanding of the control of fertility. Recent evidence suggests that E2 acts directly on GnRH neurons through estrogen receptor (ER) or a novel membrane ER (mER), as well as presynaptically through ER and mER. We have identified that inwardly rectifying K+ (Kir) conductances that are regulated by E2 play a major role in mediating GnRH cellular excitability and may be involved in negative feedback on GnRH secretion. In addition, we have discovered that the reproductively essential neuropeptide kisspeptin depolarizes GnRH neurons through inhibition of Kir channels and activation of nonselective cationic (TRPC-like) channels. The inhibition of Kir and activation of TRPC channels are important mechanisms by which kisspeptin abrogates inhibitory drive and depolarizes GnRH neurons at the time of the GnRH surge. In this proposal, we seek to further explore the mechanisms by which E2 governs GnRH neuronal excitability using whole-cell patch clamp and single cell reverse transcription PCR approaches, techniques with which we have extensive experience. We will focus on elucidating the mechanisms by which E2 modulates critical excitatory input (e.g. kisspeptin, glutamate) and inhibitory input (e.g. opioids, GABA). Our Specific Aims will examine important factors key to the understanding of GnRH excitability: (1) elucidate the signaling cascade by which E2 increases Kir channel activity in GnRH neurons; (2) elucidate the pre- and postsynaptic effects of - opioid receptor agonists on GnRH neurons; (3) elucidate E2 modulation of kisspeptin-GPR54 actions and the cellular signaling cascades activated by kisspeptin that increase TRPC channel activity in GnRH neurons; and (4) elucidate the E2 regulation of T-type calcium channels in GnRH neurons using the mER ligand STX. It is envisioned that the results from these studies will help in understanding the cellular actions of E2 that govern GnRH neuronal excitability, which is critical for pulsatile neurosecretion and ultimately ovulation in the female.